Vitamin K-2 (menatetrenone) induces iNOS in bovine vascular smooth muscle cells: No relationship between nitric oxide production and gamma-carboxylation

It has been recently reported that vitamin K-2 (menaquinone-4: menatetrenon e, VK2) has an anti-atherogenic effect as well as the ability to produce cl otting factors and improve osteoporosis. However, the mechanism by which VK 2 acts on atherosclerosis is still unclear. In this paper, we investigated the effects of vitamin K and its side chain on NO production as an anti-ath erogenic substance in a cultured vascular system. Treatment of bovine vascu lar smooth muscle cells (SMC) with VK2 (30 mu M) caused a time-dependent (2 4-72h) increase in the nitrite (NO2-) level in the conditioned medium, but not in bovine vascular endothelial cells. Classical NOS inhibitor (L-nitro arginine) and iNOS-specific inhibitors completely blocked the increased nit rite level induced by VK2 treatment, but D-nitro arginine could not it. Imm unostaining and Western blotting analysis showed that VK2 induced iNOS prot ein in the SMC. VK2 has a naphtoquinone nucleus, which is identical in mena dione (VK3), and an unsaturated side chain, which is called geranylgeraniol (GGO), To determine whether the structure of VK2 was related to an increas ing nitrite level, we investigated the nitrite level in conditioned medium treated with VK3 or GGO. Neither VK3 nor GGO treatment of SMC increased the nitrite level. In addition, warfarin, an inhibitor of VK2-dependent gamma- carboxylation, did not affect the increased nitrite level induced by VK2 in SMC, In conclusion, VK2 caused NO production through iNOS induction in bov ine SMC, that was not related to the structure of VK2, naphtoquinone nucleu s or its side chain, independently of gamma-carboxylation.